Infectious diseases are the single most common reason for physician office visits. Viruses are responsible for more of these infections than all other groups of microorganisms combined. Of all the various infections caused by viruses, the respiratory viruses (influenza A and B; parainfluenza 1, 2 and 3; respiratory syncytial virus; and adenovirus) are the most prevalent as a group. The lethality of the influenza virus was discovered in as early as 430 BC in the plague of Athens (Langmuir et al., New Engl. J. Medicine, 313 (1985) 1027). Influenza is the number one cause of acute respiratory illness and the fifth leading cause of death in the United States annually (Morbidity Mortality Weekly Report, 36 (1987) 2). As a result, the development of diagnostic methods for viruses and viral infections has become increasingly important.
The rapid diagnosis of viral infections has also become an integral part of good medical practice. Some viruses have definable antigens against which antibodies can be produced. Therefore, immunoassays have been widely used for the measurement of the presence of a virion. Where it is desirable to measure a broader group of virions, it may be possible to detect a particular component of the virus. For example, influenza viruses express surface glycoproteins having neuraminidase (sialidase) activity. The neuraminidase enzyme hydrolyzes substrates that contain 2-ketosidically linked N-acetylneuraminic acid (Neu5Ac, also known as sialic acid). Neu5Ac consists of a backbone of nine carbon atoms, a carboxyl group and an N-acetyl group. The general structure, as well as the numbering system used to denote the carbon atoms, is shown below. ##STR1## When a virion with neuraminidase activity is incubated with a chromogenic or fluorometric glycoside of Neu5Ac, the enzyme will cleave the chromogenic or fluorometric aglycon from the substrate, and the reaction product will indicate the presence of a virion.
One method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Pat. No. 5,252,458, which is incorporated herein by reference. An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al., (J. Infectious Diseases, 142 (1980) 516-523). Yolken used the 4-methylumbelliferil-2-ketoside of Neu5Ac as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated virus as well as in some nasal wash specimens from human volunteers infected with the influenza virus. Yolken suggested that "successful development of fluorometric enzyme assays for the detection of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventive and therapeutic interventions." According to Yolken, colorimetric assays were insufficiently sensitive for clinical applications. In contrast, Yolken noted that fluorometric assays might be suitable for detecting influenza neuraminidase in clinical samples.
Pachucki et al. (J. Clinical Microbiology, 26 (1988) 2664-2666) tested the 4-methylumbelliferyl-2-ketoside of Neu5Ac on clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful in detecting neuraminidase directly and rapidly in clinical specimens. The assay did, however, identify 91% virus-positive isolates 25 hours after inoculation of tissue cultures.
The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, C-4 (see above structure) seems to play an important role in enzyme-substrate interactions. Further, since it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem., 258 (1983) 12465-12471), it is essential to eliminate these undesired interactions. It has already been shown that ketosides of 4-methoxy-Neu5Ac are resistant towards bacterial sialidases, but are cleaved rapidly by viral sialidases (Beau et al., Eur. J. Biochem., 106 (1980) 531-540).
The synthesis of 4-methoxy Neu5Ac has been described (Kuhn et al., Liebigs Ann. Chem., 636 (1960) 164-173; Beau et al., Carbohydr. Res., 65 (1978) 1-10; Beau et al., Carbohydr. Res., 67 (1978) 65-77). However, the published procedure is not based on the direct alkylation of a suitably protected Neu5Ac derivative, and it involves many more steps. Also, because this method requires the use of hazardous substances such as hydrocyanic acid, it may not be commercially practical.
A direct methylation procedure is described in PCT publication WO 91/09972 (Jul. 11, 1991). According to this method, a methyl ester methyl ketoside of Neu5Ac (Neu5Ac-MEMK) is treated with tert-butyldimethylsilyl (TBMS) chloride, imidazole and a catalytic amount of 4-dimethylaminopyridine at 65.degree. C. to afford 9-O-TBMS-Neu5Ac-MEMK. Treatment of this compound with acetone and a catalytic amount of p-toluenesulfonic acid monohydrate at room temperature yields 9-O-TBMS-7,8-isopropylidene-Neu5Ac-MEMK, which is then treated with diazomethane/trifluoroborate in ether at 0.degree. C. to give the corresponding 4-methoxy derivative. This compound is deprotected by treatment with tetrabutyl-ammonium fluoride in THF, followed by alkaline hydrolysis with sodium hydroxide and acid hydrolysis with dilute hydrochloric acid/Dowex 50 (H.sup.+) to give 4-methoxy-N-acetylneuraminic acid. This method results in poor yields and requires the use of diazomethane, a gaseous reagent which is both toxic and explosive.